Phosphate-containing nanoparticle delivery vehicle

ABSTRACT

A phosphate-containing nanoparticle delivery vehicle includes a nanoparticle, an active ingredient, and a phosphodiester moiety connecting the nanoparticle and the active ingredient and forms a prodrug. The nanoparticle delivery vehicle achieves the function of increasing hydrophilicity of the active ingredient and specificity against tumor cells. Advantages of the nanoparticle material include biocompatibility, magnetism and/or controllable drug release.

RELATED APPLICATIONS

This application is a Continuation-in-part of application Ser. No.12/686,996 filed on Jan. 13, 2010 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a nanoparticle delivery vehicle, moreparticularly to a nanoparticle delivery vehicle having a phosphatemoiety.

2. Description of the Prior Art

Selective targeting of cancer cells has limited success by applicationof modern chemotherapeutic methods. Paclitaxel (i.e., Taxol) is one ofthe most popular chemotherapeutic agents used nowadays for treatment ofbreast, ovarian, and lung cancers. Being able to promote tubulinassembly into microtubules, paclitaxel brings significant impact mainlybecause of its mechanism of action. On the other hand, its drawbackscome from the lack of tumor specificity and low solubility in water.

For improving the tumor specificity and low water solubility issues ofanticancer drugs, Pero et al. (US. Patent Application No. 20030109500)administered a sufficient amount of a cytotoxic agent formulated into aphosphate prodrug form having substrate specificity for microvesselphosphatases. Microvessels therefore are destroyed preferentially overother normal tissues because the less cytotoxic prodrug form isconverted to the highly cytotoxic dephosphorylated form.

However, it may not be sufficient for highly hydrophobic anticancerdrugs to improve their hydrophilicity with single phosphate moiety, andthe hydrophilicity issue still needs to be solved. Furthermore, theabove-mentioned technique may not precisely deliver anticancer drugs tothe position of cancer cells and may not be able to selectively targetcancer cells in vivo.

To sum up, it is now a current goal to develop a novel drug deliveryvehicle for improving the hydrophilicity of anticancer drugs andprecisely delivering to the position of cancer cells.

SUMMARY OF THE INVENTION

The present invention is directed to provide a nanoparticle deliveryvehicle, which may achieve the function of increasing hydrophilicity ofthe active compound and specificity against tumor cells and providesadvantages of the nanoparticle material, such as biocompatibility,magnetism and/or controllable drug release.

A phosphate-containing nanoparticle delivery vehicle of the formula:

wherein NP is a nanoparticle; R¹ is an anticancer drug molecule; R² is amember selected from the group consisting of OH, halogen, C1-C5 alkoxygroup; each of X, Y is a member selected from the group consisting ofNH, Oand S; and Z is a member selected from the group consisting of Oand S.

Other advantages of the present invention will become apparent from thefollowing descriptions taken in conjunction with the accompanyingdrawings wherein are set forth, by way of illustration and example,certain embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the accompanying advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed descriptions,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 a is a chemical formula illustrating a nanoparticle deliveryvehicle according to an embodiment of the present invention;

FIG. 1 b is a chemical formula illustrating a nanoparticle deliveryvehicle according to an embodiment of the present invention;

FIG. 2 is a schematic diagram illustrating the preparation ofnanoparticle delivery vehicles according to an embodiment of the presentinvention;

FIG. 3 is a line chart illustrating the results of the nanoparticledelivery vehicles of the present invention;

FIGS. 4 a to 4 c are pictures illustrating the results of thenanoparticle delivery vehicles of the present invention;

FIG. 5 is a line chart illustrating the results of the nanoparticledelivery vehicles of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

A nanoparticle delivery vehicle of the present invention includes aphosphodiester moiety connecting a nanoparticle and an active ingredientto form a prodrug. The nanoparticle delivery vehicle achieves thefunction of increasing hydrophilicity of the active ingredient andspecificity against tumor cells. Advantages of the nanoparticle materialmay include biocompatibility, magnetism and/or controllable drugrelease. The design for the nanoparticle delivery vehicle of the presentinvention is described in detail as followings.

Selecting and Modifying Nanoparticles:

There are no limits on the physical parameters of a nanoparticlecomponent of the present invention. The design of a delivery vehiclemay, however, take into account the biocompatibility of the nanoparticledelivery vehicle, where appropriate. The physical parameters of ananoparticle delivery vehicle can be optimized, with the desired effectgoverning the choice of size, shape and material. Since the deliveryvehicle of the present invention would be used for carrying an activeingredient, e.g. a drug, in vivo, the biocompatibility thereof may betaken into consideration.

Among a diverse selection of nanoparticles, any of those withmagnetization, e.g. iron, cobalt, nickel and oxides thereof, may bechosen as the delivery vehicle for being detectable and tractable. Amongnanoparticles with magnetization, iron oxide nanoparticles, Fe-NPs,inherently exhibits strong magnetization and little to no toxicity invivo, and hence are preferred over the others. In the clinical field ofhuman medicine, these particles are used as delivery vehicles for drugs,genes, and radionuclides. When used to form ferrofluid, thesenanoparticles can be tracked for the purpose of contrast agents. When anexternal magnetic field is applied, these superparamagnetic Fe-NPs areallowed to be delivered to the desired target area and be fixed at aspecific site while the medication is released and acts locally.

Functionalized gold nanoparticles, Au-NPs, are promising candidates fordrug delivery because of their unique dimensions, tunablefunctionalities on the surface, and controllable drug release. Wang etal. (ChemMedChem, 2007, 2, 374-378) have revealed the application of3-mercaptopropionic acid capped Au-NPs in drug delivery and asbiomarkers of drug-resistant cancer cells.

Other biocompatible nanoparticles may also be chosen as the deliveryvehicle of the present invention, containing without limitations totitanium dioxide, zinc oxide, tin dioxide, copper, aluminum, cadmiumselenide, silicon dioxide or diamond.

The nanoparticle delivery vehicle of the present invention may befurther modified for desired properties. In one embodiment, thenanoparticle delivery vehicle of the present invention may be chemicallymodified for improved hydrophilicity. For example, these nanoparticlesmay be provided with poly-NH₃ ⁺ for improved hydrophilicity, and thepreparation thereof is referred in Yeh et al., titled “Replace WithMethod for Preparation of Water-soluble and Dispersed Iron OxideNanoparticles and Application Thereof”; Germany Patent 102004035803,2007.

In addition, these nanoparticles may be modified for increasing theirbiocompatibility as well as penetrating cell membranes. In one example,the nanoparticles may be provided with hydrophilic polyethylene glycols,PEGs, for achieving the above mechanism, and the preparation methodwould be later detailed.

Role of Phosphodiester Moiety

Chemotherapeutic agents possessing a phosphate unit would preferentiallyinteract with the cancer cells. Moreover, dephosphorylation often takesplace more easily in cancer cells than in normal cells. An advantage ofour design to incorporate the phosphodiester moiety in activeingredient-containing nanoparticles is the capability of selectivetargeting. Hydrolysis of the phosphodiester moieties with the aid ofphosphodiesterase could free active ingredients from nanoparticles.

FIG. 1 a is a schematic diagram illustrating a formula of the presentinvention, wherein the NP represents a nanoparticle, R¹ represents anactive ingredient, and NP and R¹ are coupled with a phosphodiestermoiety.

Referring to FIG. 1 b for another formula of the present invention, thephosphodiester moiety may be modified or substituted, wherein the R² maybe OH, halogen, C1-C5 alkoxy group, X, Y may be NH, O or S, and Z may beO or S.

Selecting an Active Ingredient

The active ingredient may be a drug molecule, a biological macromoleculeor a polymer. As mentioned above, chemotherapeutic agents possessing aphosphate unit would preferentially interact with the cancer cells.Moreover, dephosphorylation often takes place more easily in cancercells than in normal cells. Therefore, a preferred example of thepresent invention may be an anticancer drug molecule. The anticancerdrug molecule may include a signal transduction inhibitor, a proteaseinhibitor, an antidiabetic drug, an actin inhibitor or anantimetabolite.

Inhibitors of signal transduction pathways have been used to treatcancer resulted in reduced proliferation of cells is and/or enhancedapoptosis of tumor cells since aberrant signal transduction can lead tomalignant transformation, growth, and progression. A number of signaltransduction inhibitors (STIs) have been developed and their ability tosuppress tumor growth is currently under investigation. STIs may be usedfor targeting receptors and/or kinase belonging to a tyrosine kinase ofa platelet derived growth factor (PDGF) receptor family, an EGF receptorkinase family, an ABL kinase family, a VEGF kinase family or an srckinase family. Signal transduction inhibitors of the present inventioninclude, but are not limited to,

-   (1) insulin-like growth factor-1 (IGF-1) and insulin receptor    tyrosine kinase inhibitors such as AG 1024;-   (2) epidermal growth factor (EGF) receptor inhibitors such as    Iressa;-   (3) mTOR (mammalian target of rapamycin) inhibitors such as    Everolimus and Temsirolimus;-   (4) MEK/MAPK inhibitor such as U0126;-   (5) cyclin-dependent kinase inhibitors such as flavopiridol;-   (6) phosphatidyl inositol kinase inhibitors such as LY294002;-   (7) bcr/abl kinase inhibitors such as Gleevec; and-   (8) JAK-2 tyrosine kinase inhibitor such as AG-490.

Protease inhibitors, for example, are inhibitors to MMP (matrixmetalloproteases) which are formed to an increased extent, or areactivated, in association with diseases such as rheumatoid arthritis orcancer, thereby leading to excessive tissue breakdown, to new bloodvessels and to metastases. Target enzymes involved are, in particular,MMP-2, MMP-3 and MMP-9, as proteases, in the abovementioned pathologicalprocesses. The MMP inhibitors may include Marimastat, tetracyclines,Prinomastat, doxycycline or minocycline.

Examples of antidiabetic drugs used for anticancer purpose may includeMetformine or Phenformin. Examples of actin inhibitors may includecytochalasin B, cytochalasin D, cytochalasin A, cytochalasin E,latrunculin or Phalloidin. Examples of cytotoxic antibiotics may includeactinomycin, doxorubicin, daunorubicin, valrubicin or idarubicin.Examples of topoisomerase inhibitors may include irinotecan, topotecan,amsacrine, etoposide or teniposide. Antimetabolites may include6-Mercaptopurine (6-MP), Azathioprine (AZA), Methotrexate, 6-thioguanine(6-TG), thioguanine, 6-thioinosine or thiouridine.

In one preferred example, the drug molecule may include OH moiety forforming a phosphodiester bond. Examples of an anticancer drug having ahydroxyl group include paclitaxel, Cytarabine (Ara C), Fludarabine(Fludara®), Capecitabine (Xeloda®), Docetaxel, Epirubicin, andDoxorubicin.

In addition, small molecule drugs may include NH₂ or SH group forforming phosphodiester bond. An anticancer drug having an amine groupmay be 6-Mercaptopurine (6-MP), Azathioprine (AZA), Metformin,Phenformin or Methotrexate. An anticancer drug including thiol group maybe thiopurines such as 6-thioguanine (6-TG), thioguanine, Mercaptopurine(6-MP) and 6-thioinosine and thiouridine.

Biological macromolecules, containing without limitations to nucleicacid, nucleotide, oligonucleotide, peptide and protein, may formphosphodiester bond for delivery vehicle via their hydroxyl groups or soon.

The following descriptions of specific embodiments of the presentinvention have been presented for purposes of illustrations anddescription, and they are not intended to be exclusive or to limit thepresent invention to the precise forms disclosed.

EXAMPLE 1 Paclitaxel-Fe-NP Preparation

In one specific example of the present invention, the nanoparticle ismade of ferric oxide, which has magnetization and biocompatibility; andthe active ingredient is paclitaxel. FIG. 2 is a schematic diagramillustrating paclitaxel-conjugated nanoparticles of the presentinvention. First, the thiol terminal of tetraethylene glycol monothiol(3) was protected with a stoichiometric amount of (mono-4-methoxy)tritylchloride (MMTrCl) in the presence of triethyl amine to give(monomethoxy)tritylated thiol 2 in 65% yield. Then paclitaxel (1) wastreated with (MeO)PCl₂ (1.54 equiv) and collidine in THF,(monomethoxy)tritylated thiol 2 (1.0 equiv), I₂ (2.0 equiv), and waterin sequence to provide the desired pro-paclitaxel 4 as the major productin 72% yield.

The “one-flask method” in the conversion of 1+2→4 allowed three stepsaccomplished in situ: coupling of the paclitaxel with the PEG-SH spacer,oxidation of the phosphite center, and deprotection of the(monomethoxy)trityl group. The “one-flask method” is described in detailin Hwu, J. R et. al. (Bioorg. Med. Chem. Lett. 1997, 7, 545-548), theentire contents of which are incorporated by reference herein.

Second, we modified the ammonium groups in Fe₃O₄-nanoparticles[Fe-NP-(NH₃)+n] by using N-succinimidyl 3-(N-maleimido)propionate (1.2equiv) in DMSO to produce the functionalized Fe-NP 5. The water-solubleand dispersed Fe₃O₄-nanoparticles 5 were prepared from two solutionscontaining Fe^(II) and Fe^(III) as well as an organic acid containing anamino group. Then the pH of the solution was adjusted, and the properamount of adherent was added to achieve complete coating of the particlesurface with —NH₃ ⁺ groups.

Third, attachment of thiol 4 (4.3 equiv) to Fe-NP 5 in methanol at roomtemperature produced the desired Michael adduct paclitaxel-Fe-NP 6, ofwhich the mean diameter was 6.1±0.8 nm as determined by TEM.

Before and after conjugation, the magnetization loops of Fe-NP-(NH₃)⁺_(n) 5 and paclitaxel-Fe-NP 6 were measured at room temperature; theircurves are shown in FIG. 3. The saturation magnetization forpaclitaxel-Fe-NP 6 was determined as 4.0 emu/mg, which indicates itsmagnetic detectability and the tracking feasibility.

On the other hand, our results from thermogravimetric analysis (TGA) ofhybrid nanoparticles 5 and 6 reveal that the estimated average number ofsuccinimido linkers and paclitaxel attached on the iron oxide cores were92 and 83, respectively.

EXAMPLE 2 Hydrophilic Paclitaxel-Au-NP Preparation

Furthermore, we incorporated pro-paclitaxel 4 (500-1000 equiv) throughits thiol terminal onto colloidal Au-NPs in water at room temperature,which was prepared by reduction of HAuCl₄ with sodium citrate.

The desired hydrophilic paclitaxel-Au-NP 7 was obtained as indicated bya 19 nm hyperchromic and bathochromic shift of UV/visible peak. Thepaclitaxel-Au-NP 7 contained 201 functional paclitaxel sites on averageas determined by the TGA method. The TEM micrographs in FIG. 4 bindicates that the paclitaxel-Au-NPs 7 have a diameter of 14.6±0.7 nmwere well dispersed.

EXAMPLE 3 Hydrophobic Paclitaxel-Au-NP Preparation

While the conjugated paclitaxel-Au-NP 7 possesses good hydrophilicity,the present invention attempted to obtain hydrophobicpaclitaxel-conjugated Au-NPs 9.

Accordingly dodecanethiol ligands in the clusters 8 were exchanged withpaclitaxel-containing thiol 4 in toluene at room temperature for 120 h.The dispersed hybrid paclitaxel-conjugated Au-NPs 9, as shown in FIG. 4c, were generated with an average diameter of 2.1±0.3 nm. We determinedthe average number of the paclitaxel molecules bound on each Au-NP 9 as46 by the displacement method involving the use of mercaptoethanol.

Hydrophilicity and Biocompatibility Test

The paclitaxel-conjugated nanoparticles 6 and 7 exhibited goodhydrophilicity, of which dispersion was 312 and 288 μg/mL, respectively.In comparison with the parent paclitaxel molecule (0.4 μg/mL), theirhydrophilicity was increased 780 and 720 times. In comparison withPEG-paclitaxel 4 (3.26 μg/mL), their hydrophilicity was increased 96 and88 times.

Because the PEG linker possesses good water solubility and Fe-NP-(NH₃)⁺_(n), is miscible with water, the improvement in hydrophilicity ofpaclitaxel-Fe-NP 6 should be attributed to both the PEG spacers and theFe-NP-(NH₃)⁺ _(n), species. Our use of the flexible PEG spacer may alsooffer advantages to aid prodrugs 6 and 7 in penetrating cell membranesas well as to increase their biocompatibility.

Drug Release Test

Chemotherapeutic agents possessing a phosphate unit would preferentiallyinteract with the cancer cells. Moreover, dephosphorylation often takesplace more easily in cancer cells than in normal cells. An advantage ofour design to incorporate of the phosphodiester moiety inpaclitaxel-containing nanoparticles is their capability of selectivetargeting. Hydrolysis of the phosphodiester moieties with the aid ofphosphodiesterase could liberate free paclitaxel from nanoparticles.

The feasibility of this hypothesis was confirmed by our experiments, inwhich up to 91% of paclitaxel-containing ligand in paclitaxel-Fe-NP 6(prodrug) were hydrolyzed by phosphodiesterase after 10 days to givefree paclitaxel molecules as detected by HPLC (see FIG. 5, curve a).Paclitaxel-Fe-NP 6 therefore acts as a “biofunctional material”.

Cytotoxicity Test for Cancer Cells

Furthermore, the present invention performed an efficacy evaluation ofthe pro-drug paclitaxel-Fe-NP 6 on human cancer cells (OECM1) and humannormal cells (HUVEC) by the MTT assay. The results showed significant(i.e., 10⁴) enhancement of cytotoxicity resulting from the pro-drug tocancer cells in comparison with normal cells within 6 days. Their IC₅₀values were 5.03×10⁻⁷ and 3.58×10⁻³ μg/mL, respectively. Moreover, thereis no significant detected amount (i.e., <0.50%) of free paclitaxel 1from paclitaxel-Fe-NP 6 in FCS (calf serum, 2.50×10⁻⁴ M) after 12 days.

To sum up, the nanoparticle delivery vehicle of the present inventionincludes a nanoparticle by using Fe₃O₄ or Au as the core and aphosphodiester moiety to form a prodrug of anti-cancer drugs. Theanti-cancer drugs may be liberated in the presence of phosphodiesteraseand may also possess magnetic tracking capability and goodhydrophilicity. The nanoparticle delivery vehicle of the presentinvention may constitute a new class of candidates as anticancer drugsapplicable in many types of cancer and would be promising in clinicaldevelopment.

While the invention can be subject to various modifications andalternative forms, a specific example thereof has been shown in thedrawings and is herein described in detail. It should be understood,however, that the invention is not to be limited to the particular formdisclosed, but on the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the appended claims.

What is claimed is:
 1. A phosphate-containing nanoparticle deliveryvehicle of the formula:

wherein NP is a nanoparticle; R¹ is an anticancer drug molecule selectedfrom the group consisting of cytochalasin B, cytochalasin D,cytochalasin A, cytochalasin E, latrunculin, Phalloidin, actinomycin,doxorubicin, daunorubicin, valrubicin, idarubicin, 6-Mercaptopurine(6-MP), Azathioprine (AZA), Methotrexate, 6-thioguanine (6-TG),thioguanine, 6-thioinosine and thiouridine; R² is a member selected fromthe group consisting of OH, halogen, C1-C5 alkoxy group; each of X, Y isa member selected from the group consisting of NH, O and S; and Z is amember selected from the group consisting of O and S.
 2. Thephosphate-containing nanoparticle delivery vehicle as claimed in claim1, wherein the nanoparticle is made of metal or metallic oxide.
 3. Thephosphate-containing nanoparticle delivery vehicle as claimed in claim2, wherein the nanoparticle is made of a member selected from the groupconsisting of iron, cobalt, nickel and oxides thereof.
 4. Thephosphate-containing nanoparticle delivery vehicle as claimed in claim2, wherein the nanoparticle is made of iron oxide or gold.
 5. Thephosphate-containing nanoparticle delivery vehicle as claimed in claim1, wherein the nanoparticle is made of a member selected from the groupconsisting of titanium dioxide, zinc oxide, tin dioxide, copper,aluminum, cadmium selenide, silicon dioxide and diamond.
 6. Thephosphate-containing nanoparticle delivery vehicle as claimed in claim1, wherein the nanoparticle further comprises a polyethylene glycol(PEG).
 7. The phosphate-containing nanoparticle delivery vehicle asclaimed in claim 1, wherein X is O.
 8. The phosphate-containingnanoparticle delivery vehicle as claimed in claim 1, wherein Y is O. 9.The phosphate-containing nanoparticle delivery vehicle as claimed inclaim 1, wherein Z is O.
 10. The phosphate-containing nanoparticledelivery vehicle as claimed in claim 1, wherein R² is OH.
 11. Thephosphate-containing nanoparticle delivery vehicle as claimed in claim1, wherein the actin inhibitor is a member selected from the groupconsisting of cytochalasin B, cytochalasin D, cytochalasin A,cytochalasin E, latrunculin and Phalloidin.
 12. The phosphate-containingnanoparticle delivery vehicle as claimed in claim 1, wherein thecytotoxic antibiotic is a member selected from the group consisting ofactinomycin, doxorubicin, daunorubicin, valrubicin and idarubicin. 13.The phosphate-containing nanoparticle delivery vehicle as claimed inclaim 1, wherein the antimetabolite is a member selected from the groupconsisting of 6-Mercaptopurine (6-MP), Azathioprine (AZA), Methotrexate,6-thioguanine (6-TG), thioguanine, 6-thioinosine and thiouridine.